MCAK Activity Controls Interphase Microtubule Dynamics and Directed Cell Migration. Myers, K.A.; C. Chen, and Waterman, C.M. Directional cell migration is initiated through extracellular stimuli that coordinate changes in the cytoskeleton to establish a polarized cellular morphology. Cell polarity can be achieved through regional regulation of microtubule (MT) dynamics, including MT growth toward the leading edge and MT shortening in the cell rear. Mitotic Centromere Associated Kinesin (MCAK) is a MT depolymerase that is down-regulated in mitosis by Aurora kinase phosphorylation. While its mitotic functions have been well-characterized, whether MCAK regulates MT dynamics during cell migration is not known. We hypothesize that MCAK is down-regulated locally via a Rac1/Pak1/Aurora-A kinase signaling pathway to establish preferential MT growth toward the leading edge and to promote MT shortening within the cell rear. To test this hypothesis, we performed time-lapse imaging of fluorescently tagged EB3 as a marker of MT plus end growth in HUVEC cells and analyzed MT dynamics and cell behavior under different manipulations of the proposed signaling cascade. We find that MCAK knockdown (KD) produces expected effects on the MT cytoskeleton, including increased levels of tubulin polymer and decreased MT catastrophe frequency. MCAK-KD cells show a reduction in MT polymerization speeds and exhibit a mal-oriented MT array, as well as a statistically significant reduction in cell migration velocity, directional persistence, and distance to origin, indicating a defect in cell migration and/or polarization. These effects are rescued through expression of exogenous wild-type-MCAK, but not by expression of either an inactive (ATPase-dead) MCAK mutant or an MCAK mutant that is incapable of phospho-regulation by Aurora-A kinase. Immunolabeling of cells expressing either constitutively active-Rac1 or constitutively active-Pak1 suggests that Rac1 and Pak1 activities correlate with increased Aurora-A activity, as assayed with a phospho-specific antibody, and also correlate with decreased levels of MCAK expression. These data suggest that interphase regulation of MCAK is achieved downstream of a Rac1/Pak1/Aurora-A signaling pathway in order to locally coordinate MCAK-mediated MT depolymerization as a method to ensure proper cell polarization and motility. A manuscript describing these studies is being prepared for publication. Dr Myers received a job as a tenure-track investigator at the University of the Sciences in Philadelphia and will finis this manuscrips as an NIH special volunteer.